Luminescence assay using cyclical excitation wavelength sequence

ABSTRACT

A method for the conduct of a measurement of proximity between luminescent species based on detection of transfer of excitation energy between them. A first photoluminescent species (the “donor”) and a second photoluminescent species (the “acceptor”) are provided and are such that the donor species and the acceptor species have at least some excitation spectral regions which differ and that at least a part of the emission spectrum of the donor overlaps with at least a part of the excitation spectrum of the acceptor. The donor species is excited with a cyclical temporal sequence of wavelength bands, optionally provided as pulses or modulated in intensity, giving rise to a characteristic temporal fluctuation in emission therefrom and emission in at least one wavelength band characteristic of the acceptor is analyzed to detect the presence of the said characteristic fluctuation or a subcomponent thereof and optionally also to detect a fluctuation characteristic of direct excitation of the acceptor.

FIELD OF INVENTION

This invention describes an improved method to conduct proximitymeasurements based on the detection of transfer of excitation energybetween species labelled with luminescent chromophores.

BACKGROUND

Luminescence techniques (exemplified by, but riot limited to,fluorescence and phosphorescence) are widely used in a variety ofanalytical applications. There is much interest in using luminescentlabels as replacements for radioisotopes in assays and measurementswhere very high sensitivity is required. Luminescent labels are alsoused widely in a number of measurements where proximity between labelledspecies is to be detected. In particular, many binding assays useluminescence energy transfer measurements to detect the formation orbreakdown of a bound complex between appropriately labelled species. Themost common technique for such assays is based on a system where onecomponent of the complex (the ‘donor’) is labelled with aphotoluminescent label and a second component (the ‘acceptor’) islabelled with a species (which might or might not be luminescent) havingan absorption spectrum which overlaps with the luminescence emission ofthe first species. When the labelled species approach sufficientlyclosely a radiationless resonant transfer of energy takes place suchthat the luminescence of the excited donor species is wholly orpartially quenched while the acceptor species is excited. Radiationlesstransfer of energy of this type is only efficient when the labelledspecies approach each other within a few nanometres and the most commonexample, energy transfer between a fluorescent donor and an acceptor,has an efficiency which decreases as the sixth power of the separationbetween donor and acceptor. Proximity assays based on resonance energytransfer are very well known in the literature and have found commercialapplications, Most often the assays are conducted to detect bindingbetween labelled species, either directly (e.g., a hybridisation betweenoligonucleotides) or mediated by an analyte of interest (e.g. a‘sandwich’immunoassay), though other formats are also used. An / enzyme,for example, might be assayed on the basis of its ability to catalyseformation or breakdown of a covalent linkage between the donor andacceptor, leading to a change in the level of energy transfer betweenthem. There are very many individual combinations of labelled donor andacceptor species bearing recognition ligands such as antibodies,lectins, various peptides, proteins and glycoproteins, nucleic acids,biotin, avidin, and the like. These are used in a variety of formatswell known in the art, based for example on detection of binding of alabelled species or on competitive processes where one or more labelledspecies is displaced by an analyte.

Energy transfer assays frequently are conducted using a luminescentacceptor. detecting the sensitised emission consequent on energytransfer from the donor species. In many cases the assays preferably areconducted in a so-called homogeneous format, where the analyte isintroduced into the measurement system and the assay is conductedwithout physical separation of the bound complex. Homogeneous assays arevery convenient, and lend themselves well to automation, but there arepotential problems with this approach. There might be coloured materialspresent in the assay medium which can absorb at the wavelengths ofeither or both of the donor and acceptor species, or which can quenchluminescence from either or both of these by radiationless energytransfer. Equally, there might be other quenching species in the assaymedium. The assay medium might be luminescent, as is found in serumsamples for example where bilirubin fluorescence is common, and suchemission might overlap that of donor and/or acceptor species. Theseproblems are minimised if the sample can be diluted sufficiently toreduce effects due to coloured species and other quenchers, and if theluminescence of the donor and acceptor are distinguishable from that ofbackground. The luminescence assay must be sufficiently sensitive thatsuch dilution does not reduce signal-to-noise ratio to an unacceptabledegree.

A further problem with energy transfer assay is common to homogeneousformats and to assays where separation steps can be included. In mostassays where sensitised luminescence from the acceptor is measured,there is also some probability of direct excitation of the acceptor bythe light used to excite the donor species. This means that the acceptorfluorescence due to energy transfer is detected against a background ofdirectly excited emission which reduces the dynamic range of the assaysince small changes above background cannot readily be measured. Inadditions there might be some level of overlap between the longwavelength ‘tail’ of the emission from the donor species and thespectral region where acceptor emission is detected.

It is a purpose of the present invention to minimise the effects ofthese potential difficulties, and to provide adequate sensitivity forassay of diluted samples where necessary.

It is important to understand that sensitivity in the context ofluminescence detection is rarely limited by the ability to detect asignal. Photon counting methods are easily able to detect single atomsand molecules of fluorescent substances. It is the ability to rejectunwanted background signals that sets the limit of sensitivity for mostmeasurements, and this is a particular problem in homogeneous assayformats where many potentially luminescent species might be present inthe assay medium. Consequently, sensitivity is determined by theselectivity of detection. The present invention provides a means toincrease selectivity of detection in the context of luminescence energytransfer.

SUMMARY OF INVENTION

The invention describes an improvement in the conduct of a measurementof proximity between luminescent species based on detection of transferof excitation energy between them wherein

a first photoluminescent species (the ‘donor’) and a secondphotoluminescent species (the ‘acceptor’) are provided and are such thatthe donor species and the acceptor species have at least some excitationspectral regions which differ and that at least a part of the emissionspectrum of the donor overlaps with at least a part of the excitationspectrum of the acceptor

the donor species is excited with a cyclical temporal sequence ofwavelength bands, optionally provided as pulses or modulated inintensity, giving rise to a characteristic temporal fluctuation inemission therefrom and

emission in at least one wavelength band characteristic of the acceptoris analysed to detect the presence of the said characteristicfluctuation or a subcomponent thereof and optionally also to detect afluctuation characteristic of direct excitation of the acceptor.

Further features of the invention are defined in the appended claims.

The invention is useful in assay formats based on radiative orradiationless transfer of excitation energy between a donor and anacceptor species.

DESCRIPTION OF DRAWINGS

FIG. 1 illustrates one embodiment of apparatus for carrying out themethod of the invention; and

FIG. 2 illustrates a further embodiment of apparatus for carrying outthe method of the invention.

DESCRIPTION OF INVENTION (INCLUDING PREFERRED EMBODIMENTS)

The invention provides a means to enhance discrimination betweenemission from the acceptor species which is directly excited by theradiation used primarily to excite the donor and acceptor emission whichis sensitised by transfer of energy from the donor. The sensitisedemission from the acceptor will have the same temporal signature as tatfrom the donor whereas the directly excited emission will differ in itstemporal fluctuation.

The invention also provides improved rejection of emission fromluminescent contaminants in the sample. Detection of a characteristicfluctuation narrows the detection bandwidth relative to unfiltereddetection and allows efficient detection of the signals of interest withgood rejection of background emission.

The degree of discrimination that can be achieved will depend on therelative bandshapes of the excitation profiles for donor and acceptorspecies within the wavelength region spanned by the excitation sequence.It will often be advantageous to measure luminescence characteristic ofdirect excitation of the acceptor species in addition to sensitisedluminescence since the former can be used as an internal standard todetect and potentially to correct for effects of coloured materialsabsorbing emission from the acceptor or other quenching species thatmight be present in the assay medium. Measurements of characteristicfluctuations can also be made in a spectral region dominated by emissionfrom the donor species to determine the donor emission with suppressionof background. This is useful where assays are designed to measure theratio of donor emission to sensitised emission from acceptor, which is acommonly encountered measurement format.

In practice it will be convenient to analyse the emission fluctuation ofdonor, acceptor and background individually to determine which temporalfrequency components should be used to optimise discrimination. Thereare many methods to optimise discrimination between temporal signalsequences. For example, an optimum filter can be often be synthesisedmathematically or approximated physically according to well knownprocedures. The transfer function of a filter to separate a first andsecond signal optimally is given by the ratio of the power spectrum ofthe first signal to the sum of the power spectra of the first signal andthe second signal as described for example in Appendix 5 of ‘Electronicsfor Experimentation and Research’ (Brian K. Jones (1986), Prentice HallInternational, ISBN 0-13-2507544). It is likely that a simpler approachwill suffice in most practical circumstances. For example one or morecharacteristic fluctuation frequencies might be chosen which arerepresented strongly in the emission from one of the luminescent speciesbut which are not prominent in the directly-excited emission from otherspecies present in the assay.

The analysis means to detect the desired temporal pattern from thedetector might be one or more electronic filters or might usemathematical filtering methods on digitised data from the detector.Alternatively the temporal and/or spectral sensitivity of the detectormight be varied synchronously with the excitation sequence to effectdetection according to a predetermined correlation scheme. Measurementsof a fluctuating signal with a detector sinusoidally-modulated insensitivity can be used in conjunction with phase-sensitive detection todetermine the Fourier component in the signal at the modulationfrequency. Alternatively, the detector can be used to perform across-correlation analysis if its sensitivity is modulated according toa pattern known to be present in the signal and the result compared withthat obtained without such modulation.

The invention can be implemented using the well-known technique ofwavelength modulation, which has been used to increase selectivity inanalytical detection of fluorophores, but which has not previously beenapplied to our knowledge to proximity assay. In a wavelength modulationexperiment an excitation source is repetitively scanned across theexcitation peak of a fluorophore at a fixed frequency F. The emissionfrom the fluorophore will have a temporal component that fluctuates at2F as a consequence of the increase and decrease of emission for eachhalf cycle as the excitation scans across the peak. Any contaminatingfluorophore which has absorbance in the scanned region, but which doesnot have a peak in this region, will emit fluorescence with littletemporal fluctuation at 2F, so that a simple filter tuned to thisfrequency will increase discrimination against this source ofbackground. The principle can be extended to modulation across theemission peak maximum using a scanning optical filter or monochromatorto similar effect. If additional selectivity is required both excitationand emission can be scanned jointly at the same or different frequenciesand the resulting fluctuation pattern characteristic of the spectralbandshapes of absorption and emission can be detected by lock-in meansat an appropriate frequency or frequencies. The use of modulation inconjunction with a lock-in detection scheme has the advantage ofnarrowing the measurement bandwidth and allowing measurements to be madein one or more frequency ranges chosen to mininmise external noisecontributions.

Wavelength modulation as described is in essence a coding scheme.Luminescence from the species having a given excitation spectrum can beencoded temporally. The presence of the characteristic fluctuation inemission of the acceptor can be detected to distinguish between directlyexcited emission and sensitised emission mediated by the donor. In itsstandard form wavelength modulation does not optimise the detectedsignal however, and in addition to components at 2F much of the signalpower will be present in other tonics The method of the invention can beextended to improve on this by controlling the excitation sequence. Ifthe excitation is provided as a sequence of wavelength bands, each of apredetermined intensity, then the programmed sequence can be tailoredwith knowledge of the excitation spectrum of the target species so thatthe resultant temporal fluctuation of emission is concentrated in agiven harmonic.

The means to provide the excitation sequence of the invention might be aconventional monochromator equipped with a suitable light source. Thescanning of the monochromator is preferably controlled electronically,for example under computer control and the intensity of the light sourceis also preferably capable of programmed control. For example, a pulsedXenon flashlamp might be used with computer control of the chargingconditions to vary the light output in a programmed manner from flash toflash. Conventional optics are rather cumbersome and expensive however.The most preferred means to generate the excitation sequence is toprovide a number of light-emitting diodes or other semiconductorsources, each optionally provided with a bandpass filter to transmit aspecific wavelength region. The use of light-emitting diodes has severaladvantages.

the diodes are physically small and inexpensive

diodes are available emitting across the spectrum from the near UV tothe red

the operating lifetime of the diodes is very long in normal use

many types of diode can he pulsed and/or modulated directly at very highspeed

diodes are efficient, easily driven with low cost circuitry and generatelittle heat

diodes are easily connected to optical fibres for efficient lightdelivery or to combine light from a number of diodes.

The invention is useful in assay formats based on radiative orradiationless transfer of excitation energy between a donor and anacceptor species.

In a typical assay based on radiationless transfer of energy a donorspecies is provided in conjunction with a probe which is capable ofbinding to a target molecule. The probe might be an antigen or antibodyfor example, or might be a lectin, oligonucleotide sequence or any othermolecule capable of forming a complex with a partner. For the detectionof gene sequences by hybridisation an oligonucleotide sequence iscommonly used as probe, whereas for many other assays the tight bindingbetween an antibody and an antigen is exploited. In other cases the verystable complex formed between biotin and avidin or streptavidin is usedto mediate binding, for example using a biotinylated oligonucleotide tobind to a given gene sequence and adding a fluorescent label conjugatedto avidin or streptavidin.

Specific binding interactions are not limited to association betweenmolecules and patterned surfaces of polymer or glass can be preparedhaving a suitable shape and distribution of functional groups to bind agiven molecular species.

A common assay format suited to the invention is the ‘sandwich assay’format. An antibody capable of binding to one region of an analyte islabelled with an energy donor species and a second antibody capable ofbinding to another region of the analyte is labelled with an energyacceptor species. Ternary complexes are formed between the antibodiesand the analyte on incubation in the assay medium. Excitation of donorresults in efficient transfer of energy to the acceptor species in thecomplex because of the close proximity between donor and acceptor. Inthe present invention such energy transfer is detected by exciting thedonor species in the manner of the invention, detecting emission fromthe acceptor species and analysing the detected signal to extract thatcomponent having a fluctuation characteristic of donor emission.

Assays based on radiationless transfer of energy are limited todetection of species in close proximity. Resonance energy transfer forexample is not easily measured when donor and acceptor are separated bydistances greater than 10 nm. For some purposes this is a limitation. Insuch cases, for example when labels bind at distant sites on a DNAsequence or when an analyte is a large macromolecule and labels bind toregions more thank a few nanometres apart, the invention can be appliedusing radiative energy transfer. The requirement for radiative energytransfer is merely that a photon emitted by a donor species is absorbedby an acceptor species. This process is most probable when the acceptorspecies is in close proximity to the emitter and when the acceptor has ahigh absorbance and a large cross-section. The acceptor is thereforemost usefully provided as a highly absorbing particle containingmultiple absorbing species. Typically the acceptor species would be afluorescent particle with a size of the order of microns.

The measurement of radiative energy transfer is similar to that forradiationless energy transfer. Radiationless transfer of energy shortensthe emissive lifetime of the donor whereas radiative energy transferdoes not affect the donor's lifetime, so that measurements based on thedecay time of the donor cannot be used in the latter case. The presentinvention does not rely on measurements of the lifetime of the donorspecies and so can be applied equally to assays and measurements ofradiative and non-radiative transfer.

The invention is illustrated for example only in FIG. 1 which shows alight source (L) comprising a cluster of light-emitting diodes, eachequipped with a bandpass filter. The diodes are shown as placed aroundthe periphery of a lens so as to focus to a common point, though it isto be understood that other optical arrangements including fibre-opticdelivery could also be used. The arrangement shown is one example ofso-called ‘dark-field’ illumination, which is sometimes used in opticalmicroscopy to avoid direct light from a source of illumination fromreaching the detector. In the present context dark-field illumination isconvenient, though not essential, since it is effective in reducingbackground from optical filters in the detection lightpath. The diodesare each controlled in intensity by a driving unit (not shown) which isin turn controlled by the computer. A computer is not an absoluterequirement but is convenient as a control and analysis unit. Thecomputer furnishes a preprogrammed sequence to the driving unit whichdrives each diode with pulsed and/or modulated waveforms. A sample (S)is positioned at the common focus and is excited by the emission fromthe diodes. Emission from the sample is detected in a first detectionchannel (D) comprising an electronic detector such as a photomultiplieror semiconductor detector equipped with a wavelength selection means,shown in the figure as a bandpass filter (F) to isolate emissioncharacteristic of the acceptor species in the sample. Optionally atleast one other detection channel is provided (shown as D1) equippedwith a wavelength selection means to isolate emission characteristic ofthe donor species of the sample. Where the second channel is used inaddition to the first channel a convenient means to separate signals inthe characteristic wavelength regions is to use a beamsplitter (B). Apartially silvered mirror or more commonly a dichroic beamsplittertransmitting a first set of wavelengths to the first detection channeland reflecting a second set of wavelengths to the second channel servesthis purpose. It should be understood that other optical geometries arealso appropriate to the invention. For example a second detectionchannel can be placed at D2 as shown. Signals from the detectionchannel(s) are preconditioned by amplification and optionally byelectronic filtering before being presented to the data acquisitionunit. It should be understood that dedicated electronic signalprocessing units such as one or more lock-in amplifiers might be used toprocess signals either alone or in conjunction with the computer.

In the example the emission wavelengths are shown as being selected bybandpass filters. It can be convenient to use instead a programmablewavelength selector to allow further selectivity on the basis ofemission spectral bandshape as outlined earlier. This can be achieved byconventional means, using scanning monochromators for example. Howeverit is convenient to use solid state devices that offer compactness, highthroughput and programmable transmission efficiency. Liquid crystaltunable filters (such as are available commercially from Displaytech Ltdand Meadowlark Optics Ltd in the USA) can be scanned rapidly and offerwide apertures for efficient light collection. Acousto-optic tunablefilters (available commercially from Brimrose in the USA and Gooch andHousego in the UK, for example) can also be used as electronicallyscanned monochromators.

The invention as so far described provides an efficient means todiscriminate between directly-excited and sensitised emission from anenergy acceptor, as well as giving enhanced background rejection. Afurther consideration is the possibility of emission from the donorspecies being detected in the wavelength region characteristic of theacceptor emission. This is possible, particularly if the acceptor has asmall Stokes shift between absorption and emission (as with dyes such asfluorescein and rhodamine for example). If excitation wavelengthencoding alone is used, both donor emission and sensitised acceptoremission will have the same characteristic temporal fluctuation and willbe indistinguishable on this basis. However if the emission transmissionmeans is scanned in a programmed manner jointly with the excitationmeans, the fluctuation patterns of the donor and sensitised acceptoremission will in general be distinguishable.

The encoding scheme of the invention can also be used in conjunctionwith other means to discriminate between donor emission, backgroundemission, directly-excited acceptor emission and sensitised acceptoremission. One such means which has been described in U.S. Pat. No.4,822,733 uses donor and acceptor labels that differ in luminescencedecay time (‘lifetime’).

If a donor species with a long luminescence lifetime transfers energy toan acceptor species with a short luminescence lifetime, the donorlifetime is decreased but the sensitised emission follows the decay ofthe donor. By lifetime discrimination the directly-excited andsensitised emissions of the acceptor can be readily distinguished. Thisprinciple is used in a commercial range of binding assays marketed byPackard Bioscience where long-lived lanthanide species transferexcitation energy to short-lived phycobiliproteins for example. Althoughlifetime resolution is effective in discriminating between directexcitation of acceptor and sensitised excitation from the donor, it doesnot discriminate effectively against other sources of long-livedemission that arise from contaminants in the assay medium. Other sourcesof long-lived luminescence are phosphorescence from microplates used inmany assay protocol and luminescence emission from optical filters. Thusthe method of the present invention can advantageously be combined withlifetime resolution to minimise these sources of background. Forefficient operation it is necessary for lifetimes of species to bedistinguished to differ appreciably, and in practice at least by afactor of three or very preferably more. If the encoding scheme uses atunable emission filter to induce characteristic fluctuations dependenton the emission bandshape of the labels, it is also possible to minimisecontributions from unwanted long-lived donor emission ‘leaking’ into theacceptor spectral emission band.

Suitable long-lived donor species amenable to wavelength encoding arecomplexes of ruthenium with ligands such as bipyridine andbathophenanthroline. These are excited with blue light and can transferenergy to dyes absorbing near 600 nm and emitting further in the redsuch as phycobiliproteins. It is desirable to use labels which can beexcited efficiently by light of wavelength longer than c. 380 nm sincethis is close to the present short wavelength limit of emission fromlight-emitting diodes.

An alternative means to implement an assay using lifetime resolution wasdisclosed in U.S. Pat. No. 4,822,733, but has not found widespread use.This is a format where the donor species is short lived while theacceptor has a longer fluorescence lifetime. As described, this approachsuffers from the serious disadvantage that directly-excited acceptorluminescence is indistinguishable from that sensitised by the donor,since both are long-lived. It does allow efficient rejection ofscattered light and short-lived background emission however. There is afurther advantage that a large number of potential donors are known asshort-lifetime species but there are fewer long-lived labels suitablefor use as energy donors. As an example, a variety of types of labelledmicrosphere are suitable for use as energy acceptors and binding ofsmall molecules to such microspheres can readily be detected by energytransfer to the multiple absorbing species of the particle. Thesupporting matrix minimises quenching and allows many dyes to givelong-lived emission. However such labels are inefficient as energydonors to small molecules unless the particles are extremely small.

The present invention overcomes the difficulty inherent in the use of along-lived acceptor with a short lived donor, Both the directly-excitedand sensitised luminescence from the acceptor will be long-lived, butthe excitation-wavelength encoding process will allow thesecontributions to be distinguished. As examples of suitable energytransfer pairs, dyes such as fluorescein can transfer to long-livedlabels such as eosin or phthalocyanines bound to polymer particles or inglassy matrices. Blue-green emitting dyes such as coumarins excited inthe blue, violet and near ultra-violet regions could be used to transferto long-lived metal complexes such as ruthenium bipyridyl complexes. Inthese cases the directly excited emission serves as an internal standardto detect quenching of the lone-lived emitter.

The combination of fluorescence lifetime discrimination with an encodedwavelength sequence for excitation and/or emission is also potentiallyvaluable as a means to reduce background in the detection of fluorescentlabels where energy transfer is not under investigation. For example andnot limitation, the label ethidium bromide binds to DNA and itsfluorescence lifetime and absorption maximum both differ between boundand free forms of the dye. Both of these effects can be combined toallow selective detection of bound dye with rejection of backgroundfluorescence from free dye and from other fluorescent materials in thesample. This increase in selectivity increases signal-to-noise ratio andhence improves detection sensitivity for DNA and related molecules usingthe dye.

Fluorescence lifetime discrimination can be combined with excitationtemporal coding in a number of ways. One approach uses light-emittingdiodes as the excitation source. Many of these diodes are capable of usein a pulsed mode with pulse widths down to nanoseconds, and can also bemodulated in intensity at very high speed. An array of such diodes, eachequipped with filters to isolate a given wavelength band and chosen tocover an appropriate spectral range, forms a very appropriate excitationsource for the present invention. It is straightforward to drive thediodes in a preprogrammed sequence with fast or slow pulses or to drivethe diodes with continuous waveforms such as sinusoids. It can beconvenient to link such diodes to optical fibres which can be combinedinto a bundle, for example as randomised mixtures of fibres from eachdiode, to facilitate use with other optical systems. In someapplications, particularly where it is required to generate UV light andwhere high pulsed currents are required, it can be necessary to coot thediode structures, and this is conveniently achieved by the use ofthermoelectric coolers based on the Peltier effect.

An example where lifetime resolution is combined with spectral encodingis given in FIG. 2. This describes a system where several light-emittingdiodes (shown in the Figure as three diodes (L)), each equipped withoptical filters to isolate a different range of excitation wavelengthsare used to illuminate a common region of a sample (S) containing donorand acceptor species differing in lifetime of luminescence which isviewed by a photomultiplier (P) equipped with an optical filter (F) toisolate the emission from a component of interest in the sample. Thediodes are energised in sequence giving rise to characteristicfluctuations in emission from the sample component, and the pulseduration and intensity from each diode is determined by the control unit(CON). The signal from the detector is amplified in a fast amplifier(A), the output of which passes to a gating unit (G) with apredetermined delay and gate width, and the output of this unit is fedto a signal processing unit (C1). The output of amplifier (A) alsopasses directly into a similar signal processing unit (C2). These unitscomprise programmable filters or correlators able to isolate frequencycomponents characteristic of the temporal fluctuation in emission due tothe sequence of excitation wavelengths illuminating the sample. Thecontrol unit driving the diodes is programmed via a computer (COMP)which also serves to control the detector delay and gating unit (G) andthe processing units (C1) and (C2). The output signal from unit (C1)corresponds to long-lived emission from the sample that also has thetemporal fluctuation detected by (C1), while that from (C2) is sensitiveto prompt emission in addition to longer-lived emission. It should beclear that the example can be varied and extended in a variety of waysand serves only to illustrate the operation of the invention.

Some light emitting diodes have a spectral emission which varies inresponse to temperature and/or to drive current. Such diodes can be usedfor the present invention by providing them with programmed changes intemperature and/or with programmed sequences of drive current of varyingintensity and duration. The wavelength encoding is thus implemented bythe sequence in which the diodes are driven, while the fast lifetimeresolution is implemented by gated detection or by frequency domainmethods well known in the art. As an alternative the excitation sourcemight provide two or more wavelengths simultaneously, each coded with adistinguishable temporal sequence of intensity.

Light-emitting diodes presently available are not able to generatewavelengths much below 380 nm efficiently and an alternative approach isrequired to implement short wavelength excitation. One approach is touse a set of deuterium light sources. These are very stable and generatea continuous spectrum of light down to below 200 nm. Deuterium sourcescan be modulated at radio frequency and can be pulsed at high speed. Wehave described the use of deuterium sources for fluorescence lifetimemeasurement in ‘A compact frequency-domain fluorometer with adirectly-modulated deuterium light source’, by C. G. Morgan, Y. Hua, A.C. Mitchell, J. G. Murray and A. D. Boardman, (Review of ScientificInstruments vol. 67, no.1, pages 41-47, (1996)) and in a related patentFP-A-0 519 930. Although the deuterium source is particularly suited tothe present invention where short wavelength excitation is required, itshould be apparent that other discharge sources might also be used. Forexample, mercury vapour can be excited to give pulses of ultra-violetlight though the minimum pulse width is longer than for a deuteriumsource because of the nature of the electronic transitions concerned.Xenon sources can generate short pulses of visible and UV light and acommercial Xenon flashlamp from IBH Ltd in the UK can provide pulsewidths as low as 100 ns, though with a small percentage of longer-livedafterglow. Hydrogen discharges are capable of very fast pulsedoperation, but are less intense than deuterium sources operated undersimilar conditions. Many other elements can be included in dischargetubes, and give rise to characteristic emissions that can be used forexcitation in the present invention.

For the present invention it is convenient to drive a number ofoptically filtered sources each with fast pulses or alternatively tocode each source with a different frequency or set of frequencies sothat their contributions to the excitation process can be decodedsubsequently.

As an alternative to single-photon excitation, it is known that manyluminescent materials can be excited by simultaneous or sequentialabsorption of several long wavelength photons to achieve an excitedstate of higher energy than that of any individual photon used forexcitation. This process could also be used in the present invention. Inthis case the detected pattern is that due to the variation oftwo-photon or multiphoton excitation cross-section across the absorptionband of the donor as different wavelengths or combinations of Wavelengthare provided in sequence to match energy levels across the single-photonabsorption band. This is to be distinguished from schemes wheremultiphoton excitation is limited to a particular combination ofwavelengths and is not associated with exploitation of spectralvariations of donor cross-section for temporal coding. Examples ofmultiphoton excitation of fluorophores are given in EP-A-0 666 473 andU.S. Pat. No. 5,034,613 and labels suitable for multiphoton excitationare discussed in WO-A-94/07142 (PCT/US93/08712) and in our co-pendingpatent application WO-A-99/43072 (PCT/GB98100/769). Suitable labels arecommonly particles or crystals containing a number of emissive specieswithin the protective matrix, and often in conjunction with sensitisingagents that can transfer energy to the emissive species. For exampleupconversion phosphors are known based on ytterbium ion as sensitisertransferring energy by a multistep process to luminescent lanthanideions such as erbium and thulium. Upconversion is efficient for ions in amatrix characterised by a low phonon energy to minimise radiationlessdeactivation of the excited species.

The detectors for the present invention can be of any type, but theinvention is well suited to use with imaging detectors such as CCDarrays, intensified imaging detectors and the like. Some versions ofsuch detectors can also be gated or modulated to implementlifetime-resolved detection with nanosecond resolution or better. Forexample, image intensifiers can be used in this way as described in ourpatent EP-A-0 519 930, while interline CCD cameras can be electronicallyshuttered or can be used in a modulated form. Modulated CCD imagerssuitable for lifetime-resolved imaging in the microsecond time range areavailable commercially from Photonic Research Systems Ltd in the UK.Electronic shuttering of CCDs has also been investigated by this companyand time resolution of a few nanoseconds has been achieved.

Imaging detectors offer the possibility of making large numbers ofmeasurements in parallel and are well suited to assay procedures such ashigh-throughput drug screening and other areas where the presentinvention finds application. Imaging detection as outlined also offersthe means to combine lifetime resolution with spectral encoding schemesof the present invention for simultaneous measurements on severalsamples and this is an important advantage.

Imaging detectors can be used with coded excitation sequences, eitherdecoding the signal by serially processing a sequence of images, or elseby interposing an optical modulator such as a controllable light valveor attenuator between the sample and the detector. Alternatively, insome cases the sensitivity of the detector can be modulated directly asdescribed above, There are also available options for decoding a signalbased on the emission spectral bandshape, for example using an imagingacousto-optic tunable filter, electrically-tunable liquid-crystalwavelength filter or tunable Fabry-Perot cavity. Interference filterscan also be used as tunable elements if they are tilted from the opticalaxis. Other filters have been described in the literature and may besuitable for the purposes of the invention. For example a so-calledChristiansen filter can be implemented by dispersing particles of onemedium in a second medium of different refractive index, where therefractive index of one component varies markedly with wavelength andthat of the other medium varies with temperature. The filter is tuned byvariation in temperature and transmits light at a wavelength where therefractive index of the medium matches that of the dispersed phase.Variants on the principle can be considered where the refractive indexof one of the components can be tuned electrically (e.g. a liquidcrystal) or via pressure variation.

What is claimed is:
 1. A method of effecting measurement of proximitybetween luminescent species based on detection of transfer of excitationenergy therebetween, the method comprising providing a firstphotoluminescent species (the ‘donor’) and a second photoluminescentspecies (the ‘acceptor’) which are such that the donor species and theacceptor species have at least some excitation spectral regions whichdiffer and also such that at least a part of the emission spectrum ofthe donor overlaps with at least a part of the excitation spectrum ofthe acceptor, exciting the donor species with an excitation source, andanalyzing emission of the acceptor using a detection means,characterized in that the donor species is excited with a cyclicaltemporal sequence of wavelength bands, optionally provided as pulses ormodulated in intensity, giving rise to a characteristic temporalfluctuation in emission from the donor species, and emission in at leastone wavelength band characteristic of the acceptor is analyzed to detectthe presence of the said characteristic fluctuation or a subcomponentthereof and optionally also to detect a fluctuation characteristic ofdirect excitation of the acceptor.
 2. A method according to claim 1wherein luminescence is also measured in a spectral regioncharacteristic of donor emission and a temporal fluctuationcharacteristic of the donor is detected.
 3. A method according to claim1 wherein excitation wavelengths are scanned cyclically in a continuousor discrete manner across the excitation maximum of the donor at a firstscanning frequency and luminescence is detected at one temporalfrequency or more that is an integral multiple of the said scanningfrequency.
 4. A method according to claim 3 wherein the excitationwavelengths are each programmed in intensity so as to concentratedetected signal in a given harmonic of the cyclic scanning frequency. 5.A method according to claim 1 wherein the excitation source is aplurality of light-emitting diodes, operated individually or in groupsin a programmed sequence and with a programmed intensity.
 6. A methodaccording to claim 1 wherein the excitation source comprises a pluralityof light sources coupled to a fibre-optic light guide or liquid lightguide.
 7. A method according to claim 6 wherein the light sources arebased on plasma emission from excited hydrogen, deuterium, mercury,xenon or other element.
 8. A method according to claim 1 wherein theexcitation source illuminates a sample in a ‘dark-field’ configurationsuch that the sample is excited obliquely or with a hollow cone of lightilluminating a given region in space and wherein the said region isviewed by one or more detection means at an angle or angles chosen tominimize direct exposure of the said detection means to the excitingradiation.
 9. A method according to claim 1 wherein the detection meansis equipped with a tunable wavelength-selective filter and this isscanned cyclically across at least a part of the emission band of theacceptor species giving rise to a characteristic temporal fluctuation indetected signal therefrom, and the superposition of the said fluctuationand that due to the cyclic variation of excitation wavelength ismeasured to detect the joint fluctuation or characteristic temporalcomponents thereof.
 10. A method according to claim 1 wherein thedetection means is equipped with a tunable optical filter comprising orcontaining a liquid-crystalline medium, an acousto-optic tunable filter,a tunable Fabry-Perot cavity filter, an interference filter tuned bytilting from the optical axis or a filter based on wavelength-dependentscattering from particles in a medium, such as a Christiansen filter.11. A method according to claim 1 wherein the detection means is animaging detector.
 12. A method according to claim 11 wherein thedetection means can be gated or modulated in sensitivity and where suchgating or modulation is used to facilitate selective detection ofemission from one or more of the donor and acceptor.
 13. A methodaccording to claim 1 wherein the excitation of the donor species is bysimultaneous or sequential absorption of two or more photons of the sameor different wavelength.
 14. A method according to claim 13 wherein thedonor species is an upconversion medium based on direct or sensitizedexcitation of emissive species within a supporting matrix.
 15. A methodaccording to any preceding claim wherein the excitation means is drivento produce pulsed or modulated light and a measurement is made bytime-domain or frequency-domain methods to detect a signal jointly onthe basis of the emissive lifetime of one or more of the donor andacceptor and of the temporal fluctuations in detected emission resultingfrom either or both of the cyclic variation of excitation wavelength andthe cyclic variation of optical wavelengths passed to the detector. 16.A method according to claim 15 wherein the donor has an emissivelifetime at least threefold longer than that of the acceptor.
 17. Amethod according to claim 15 wherein the acceptor has an emissivelifetime at least threefold longer than the donor.